The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

miR-602 Mediates the RASSF1A/JNK Pathway, Thereby Promoting Postoperative Recurrence in Nude Mice with Liver Cancer.

PURPOSE: At present, there are few studies on the mechanisms underlying postoperative recurrence of liver cancer, and the mechanism of action of miR-602 in postoperative recurrence of liver tumors is not clear. Our goals were to investigate the effects of miR-602 on the expression of the Ras-associated domain family 1A (RASSF1A) gene and the regulation of primary and recurrent hepatic tumors to clarify the molecular mechanisms of miR-602 in postoperative hepatocellular carcinoma. METHODS: We constructed a mouse liver orthotopic tumor model and a mouse liver recurrent tumor model. We measured the expression levels of the RASSF1A gene and then analyzed the effects of miR-602 on the regulation of RASSF1A. We transiently transfected the miR-602 gene into cells that stably overexpressed RASSF1A and examined relevant indicators to elucidate the mechanisms by which miR-602 regulates the RASSF1A/c-Jun N-terminal kinase (JNK) pathway in recurrence and dormancy in liver cancer. RESULTS: RASSF1A expression was inversely related to that of JNK, activating transcription factor 2 (ATF-2), and c-Jun in SMMC7721 cells stably transfected with the RASSF1A gene and in recurrent mouse tumor tissues. After transient transfection of cells with miR-602 mimic or miR-602 inhibitor, the expression of miR-602 was inversely related to that of RASSF1A. CONCLUSION: MiR-602 might inhibit the JNK signaling pathway by inhibiting the expression of RASSF1A, thereby promoting recurrence of liver cancer after surgery. The low expression levels of miR-602 in liver cancer tissues were closely related to postoperative recurrence; they could be used as a marker to judge the prognosis of patients with liver cancer.

Jianpi Huayu Decoction Attenuates the Immunosuppressive Status of H22 Hepatocellular Carcinoma-Bearing Mice: By Targeting Myeloid-Derived Suppressor Cells.

Tumor-induced immunosuppressive microenvironment in which myeloid-derived suppressor cells (MDSCs) plays an important role, remains an obstacle for effective oncotherapy currently. Inducing MDSCs into maturation was confirmed as an effective method to reduce the tumor-bearing host's immunosuppression. Traditional Chinese medicines (TCM) possess characteristics of alleviating immunosuppression of cancer patients and low toxicity. Jianpi Huayu Decoction (JHD) was an experienced formula of TCM for oncotherapy based on TCM theory and clinical practice. We previously observed that JHD attenuated the expression of interleukin-10 (IL-10) and transforming growth factor beta (TGF-ß) in tumor. IL-10 and TGF-ß were found to be cytokines positively related to immunosuppression induced by MDSCs. Here, our study was designed to further investigate the regulation of JHD on the immune system in the H22 liver-cancer mouse model. Mainly, flow cytometry was used to detect the proportion of immune cells, to analyze the apoptosis, differentiation and reactive oxygen species of MDSCs. We found that JHD significantly reduced the destruction of spleen structure, reduced the proportion of regulatory T cells (Treg) and T helper 17 cells (Th17), and increased the proportion of cytotoxic T lymphotes (CTL), Dendritic cells (DC) and CD11b+Gr-1+cells in spleen, but with no significant change of T helper 1 cells (Th1), T helper 2 cells (Th2) and macrophages. In vitro experiments showed that apoptosis of MDSCs was decreased as the time of JHD stimulation increased, which partly explained the increase of CD11b+Gr-1+cells in the spleen. Meanwhile, JHD could promote the differentiation of MDSCs into macrophages and dendritic cells, attenuate expression of ROS in MDSCs and reduce its inhibition on the proliferation of CD4+ T cells, in vitro. Therefore, that the proportion of CD11b+Gr-1+ cells increased in the spleen of tumor-bearing hosts may not be villainy after treatment, when these drugs suppress the immunosuppressive ability of CD11b+Gr-1+ cells and promote it mature to replenish dendritic cell, at the same time. Generally, JHD may be a complementary and alternative drug for attenuating the immunosuppressive status induced by hepatocellular carcinoma, possibly by promoting differentiation and inhibiting the immunosuppressive activity of MDSCs.

Jianpi-Huayu Formula Inhibits Development of Hepatocellular Carcinoma by Regulating Expression of miR-602, Which Targets the RASSF1A Gene.

The traditional Chinese medicine formula Jianpi-Huayu (JPHY) has been reported to be effective in the treatment of hepatocellular carcinoma (HCC). However, its underlying mechanism remains unclear. In this article, we employed an orthotopic transplantation model in nude mice to explore whether JPHY could inhibit the development of HCC by regulating miR-602, which targets the Ras association domain-containing protein 1A (RASSF1A) pathway. HCC SMMC-7721 cells were treated with JPHY to test whether the RASSF1A gene as mediated by miR-602 affected the proliferation and apoptosis of tumor cells. We subsequently detected miR-602, RASSF1A, and tumor cell apoptosis-related markers in cells and liver tumor tissues. We observed that mice treated with JPHY had smaller tumors and higher survival rates than untreated ones. Similarly, JPHY-treated SMMC-7721 cells exhibited alterations in morphology and higher cytotoxicity compared with the control group. Furthermore, we found that JPHY decreased overexpression of miR-602 and increased protein expression levels of the RASS1A gene, which in turn altered protein expression levels of tumor cell apoptosis-related genes in the cells and liver tumor tissues of drug-treated mice. These results indicated that JPHY could potentially be used to treat HCC by targeting miR-602, which targets the RASSF1A gene, which in turn plays a major role in HCC pathogenesis.

TNF-α derived from M2 tumor-associated macrophages promotes epithelial-mesenchymal transition and cancer stemness through the Wnt/ß-catenin pathway in SMMC-7721 hepatocellular carcinoma cells.

M2-polarized tumor-associated macrophages (M2-TAMs) infiltrating the tumor microenvironment contribute to hepatocellular carcinoma (HCC) progression. It was reported that cancer cells undergoing EMT will acquire stemness characteristics. Here, the HCC SMMC-7721 cell line was co-cultured with M2-TAMs polarized from THP-1 cells in vitro. In in vivo studies, we used nude mice subcutaneous tumor model to test whether the growth of the tumor was affected by M2-TAMs. Subsequently, EMT, stemness and Wnt/ß-catenin pathway related markers were detected in cells and subcutaneous tumor tissues. TNF-α was also assessed in both the co-culture system supernatants and in nude mice serum. We found that SMMC-7721 underwent EMT and acquired stemness after co-culture with M2-TAMs, and resulted in larger tumor size following subcutaneous injection of SMMC-7721 suspended in M2-TAMs supernatants compared with SMMC-7721 alone. Enzyme linked immunosorbent assay showed that TNF-α expression was elevated in supernatants of M2-TAMs and positively correlated with tumor size in the serum of nude mice. Furthermore, we found that the Wnt/ß-catenin pathway was a downstream target of TNF-α and that the Wnt/ß-catenin inhibitor ICG-001 partially reversed EMT and attenuated cancer stemness. Our results indicate that TNF-α derived from M2-TAMs promote EMT and cancer stemness cells via the Wnt/ß-catenin pathway.

Tumor-associated macrophage-derived cytokines enhance cancer stem-like characteristics through epithelial-mesenchymal transition.

Cancer stem cells are a small population of cells with the potential for self-renewal and multi-directional differentiation and are an important source of cancer initiation, treatment resistance, and recurrence. Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their epithelial phenotype and convert to mesenchymal cells. Recent studies have shown that cancer cells undergoing EMT can become stem-like cells. Many kinds of tumors are associated with chronic inflammation, which plays a role in tumor progression. Among the various immune cells mediating chronic inflammation, macrophages account for ~30%-50% of the tumor mass. Macrophages are highly infiltrative in the tumor microenvironment and secrete a series of inflammatory factors and cytokines, such as transforming growth factor (TGF)-ß, IL-6, IL-10, and tumor necrosis factor (TNF)-α, which promote EMT and enhance the stemness of cancer cells. This review summarizes and discusses recent research findings on some specific mechanisms of tumor-associated macrophage-derived cytokines in EMT and cancer stemness transition, which are emerging targets of cancer treatment.